Depletion of tumor suppressor Kank1 induces centrosomal amplification via hyperactivation of RhoA.

Chromosome instability, frequently found in cancer cells, is caused by a deficiency in cell division, including centrosomal amplification and cytokinesis failure, and can result in abnormal chromosome content or aneuploidy. The small GTPase pathways have been implicated as important processes in cell division. We found that knockdown of a tumor suppressor protein Kank1 increases the number of cells with a micronucleus or bi-/multi-nuclei, which was likely caused by centrosomal amplification. Kank1 interacts with Daam1, known to bind to and activate a small GTPase, RhoA, in actin assembly. Knockdown of Kank1 or overexpression of Daam1, respectively, hyperactivates RhoA, potentially leading to the modulation of the activity of Aurora-A, a key regulator of centrosomal functions, eventually resulting in centrosomal amplification. Kank1 is also associated with contractile ring formation in collaboration with RhoA, and its deficiency results in the interruption of normal daughter cell separation, generating multinucleate cells. Such abnormal segregation of chromosomes may cause further chromosomal instability and abnormal gene functions, leading to tumorigenesis. Thus, Kank1 plays a crucial role in regulating the activity of RhoA through retrieving excess Daam1 and balancing the activities of RhoA and its effectors.

DNA microarray-based gene expression profiling of estrogenic chemicals.

We summarize updated information about DNA microarray-based gene expression profiling by focusing on its application to estrogenic chemicals. First, estrogenic chemicals, including natural/industrial estrogens and phytoestrogens, and the methods for detection and evaluation of estrogenic chemicals were overviewed along with a comprehensive list of estrogenic chemicals of natural or industrial origin. Second, gene expression profiling of chemicals using a focused microarray containing estrogen-responsive genes is summarized. Third, silent estrogens, a new type of estrogenic chemicals characterized by their estrogenic gene expression profiles without growth stimulative or inhibitory effects, have been identified so far exclusively by DNA microarray assay. Lastly, the prospect of a microarray assay is discussed, including issues such as commercialization, future directions of applications and quality control methods.

Fluorescence-based bioassays for the detection and evaluation of food materials.

We summarize here the recent progress in fluorescence-based bioassays for the detection and evaluation of food materials by focusing on fluorescent dyes used in bioassays and applications of these assays for food safety, quality and efficacy. Fluorescent dyes have been used in various bioassays, such as biosensing, cell assay, energy transfer-based assay, probing, protein/immunological assay and microarray/biochip assay. Among the arrays used in microarray/biochip assay, fluorescence-based microarrays/biochips, such as antibody/protein microarrays, bead/suspension arrays, capillary/sensor arrays, DNA microarrays/polymerase chain reaction (PCR)-based arrays, glycan/lectin arrays, immunoassay/enzyme-linked immunosorbent assay (ELISA)-based arrays, microfluidic chips and tissue arrays, have been developed and used for the assessment of allergy/poisoning/toxicity, contamination and efficacy/mechanism, and quality control/safety. DNA microarray assays have been used widely for food safety and quality as well as searches for active components. DNA microarray-based gene expression profiling may be useful for such purposes due to its advantages in the evaluation of pathway-based intracellular signaling in response to food materials.

Effects of brefeldin A-inhibited guanine nucleotide-exchange (BIG) 1 and KANK1 proteins on cell polarity and directed migration during wound healing.

Brefeldin A-inhibited guanine nucleotide-exchange protein (BIG) 1 activates class I ADP ribosylation factors (ARFs) by accelerating the replacement of bound GDP with GTP to initiate recruitment of coat proteins for membrane vesicle formation. Among proteins that interact with BIG1, kinesin family member 21A (KIF21A), a plus-end-directed motor protein, moves cargo away from the microtubule-organizing center (MTOC) on microtubules. Because KANK1, a protein containing N-terminal KN, C-terminal ankyrin-repeat, and intervening coiled-coil domains, has multiple actions in cells and also interacts with KIF21A, we explored a possible interaction between it and BIG1. We obtained evidence for a functional and physical association between these proteins, and found that the effects of BIG1 and KANK1 depletion on cell migration in wound-healing assays were remarkably similar. Treatment of cells with BIG1- or KANK1-specific siRNA interfered significantly with directed cell migration and initial orientation of Golgi/MTOC toward the leading edge, which was not mimicked by KIF21A depletion. Although colocalization of overexpressed KANK1 and endogenous BIG1 in HeLa cells was not clear microscopically, their reciprocal immunoprecipitation (IP) is compatible with the presence of small percentages of each protein in the same complexes. Depletion or overexpression of BIG1 protein appeared not to affect KANK1 distribution. Our data identify actions of both BIG1 and KANK1 in regulating cell polarity during directed migration; these actions are consistent with the presence of both BIG1 and KANK1 in dynamic multimolecular complexes that maintain Golgi/MTOC orientation, differ from those that might contain all three proteins (BIG1, KIF21A, and KANK1), and function in directed transport along microtubules.

Potential involvement of KANK1 haploinsufficiency in centrosome aberrations.

KANK1 was found as a tumor suppressor gene based on frequent deletions in renal cell carcinoma and the inhibitory activity of tumor cell proliferation. Previously, we reported that knockdown of KANK1 induced centrosomal amplification, leading to abnormal cell division, through the hyperactivation of RhoA small GTPase. Here, we investigated the loss of KANK1 function by performing CRISPR/Cas9-based genome editing to knockout the gene. After several rounds of genome editing, however, there were no cell lines with complete loss of KANK1, and the less the wild-type KANK1 dosage, the greater the number of cells with abnormal numbers of centrosomes and rates of cell-doubling and apoptosis, suggesting the involvement of KANK1 haploinsufficiency in centrosome aberrations. The rescue of KANK1-knockdown cells with a KANK1-expressing plasmid restored the rates of cells exhibiting centrosomal amplification to the control level. RNA-sequencing analysis of the cells with reduced dosages of functional KANK1 revealed potential involvement of other cell proliferation-related genes, such as EGR1, MDGA2, and BMP3, which have been reported to show haploinsufficiency when they function. When EGR1 protein expression was reduced by siRNA technology, the number of cells exhibiting centrosomal amplification increased, along with the reduction of KANK1 protein expression, suggesting their functional relationship. Thus, KANK1 haploinsufficiency may contribute to centrosome aberrations through the network of haploinsufficiency-related genes.

Estrogenic Prenylated Flavonoids in Sophora flavescens.

Sophora flavescens is a medicinal herb distributed widely in Japan and it has been used to treat various diseases and symptoms. To explore its pharmacological use, we examined the estrogenic activity of four prenylated flavonoids, namely kurarinone, kushenols A and I, and sophoraflavanone G, which are characterized by the lavandulyl group at position 8 of ring A, but have variations in the hydroxyl group at positions 3 (ring C), 5 (ring A) and 4' (ring B). These prenylated flavonoids were examined via cell proliferation assays using sulforhodamine B, Western blotting, and RT-PCR, corresponding to cell, protein, and transcription assays, respectively, based on estrogen action mechanisms. All the assays employed here found weak but clear estrogenic activities for the prenylated flavonoids examined. Furthermore, the activities were inhibited by an estrogen receptor antagonist, suggesting that the activities were likely being mediated by the estrogen receptors. However, there were differences in the activity, attributable to the hydroxyl group at position 4', which is absent in kushenol A. While the estrogenic activity of kurarinone and sophoraflavanone G has been reported before, to the best of our knowledge, there are no such reports on kushenols A and I. Therefore, this study represents the first report of their estrogenic activity.

Estrogen-induced cell signaling in the sexually dimorphic nucleus of the rat preoptic area: potential involvement of cofilin in actin dynamics for cell migration.

Estrogen is a key factor to induce the sexually dimorphic nucleus (SDN) in the preoptic area (POA) of the rat brain. Identification of estrogen-dependent signaling pathways at SDN in POA during the critical period is a prerequisite for elucidating the mechanism. In the present study, we treated female rats with/without 17ß-estradiol (E2) at birth, designated as postnatal day 1 (P1), and prepared total RNA from brain slices containing SDN for DNA microarray analysis. Among the estrogen-responsive genes identified, protein kinase C-delta (PKC-δ) was significantly up-regulated by E2 at P5. We examined the downstream effectors of PKC-δ protein by Western blotting and found an E2-induced PKC-δ/Rac1/PAK1/LIMK1/cofilin pathway. In the pathway, E2 suppressed the phosphorylation (inactive form) of cofilin. This result was supported by immunohistochemistry, where the phosphorylation/dephosphorylation of cofilin occurred at SDN, which suggests that cell migration is a cue to create sexual dimorphism in POA.

Estrogenic flavonoids and their molecular mechanisms of action.

Flavonoids are a major group of phytoestrogens associated with physiological effects, and ecological and social impacts. Although the estrogenic activity of flavonoids was reported by researchers in the fields of medical, environmental and food studies, their molecular mechanisms of action have not been comprehensively reviewed. The estrogenic activity of the respective classes of flavonoids, anthocyanidins/anthocyanins, 2-arylbenzofurans/3-arylcoumarins/α-methyldeoxybenzoins, aurones/chalcones/dihydrochalcones, coumaronochromones, coumestans, flavans/flavan-3-ols/flavan-4-ols, flavanones/dihydroflavonols, flavones/flavonols, homoisoflavonoids, isoflavans, isoflavanones, isoflavenes, isoflavones, neoflavonoids, oligoflavonoids, pterocarpans/pterocarpenes, and rotenone/rotenoids, was summarized through a comprehensive literature search, and their structure-activity relationship, biological activities, signaling pathways, and applications were discussed. Although the respective classes of flavonoids contained at least one chemical mimicking estrogen, the mechanisms varied, such as those with estrogenic, anti-estrogenic, non-estrogenic, and biphasic activities, and additional activities through crosstalk/bypassing, which exert biological activities through cell signaling pathways. Such mechanistic variations of estrogen action are not limited to flavonoids and are observed among other broad categories of chemicals, thus this group of chemicals can be termed as the "estrogenome". This review article focuses on the connection of estrogen action mainly between the outer and the inner environments, which represent variations of chemicals and biological activities/signaling pathways, respectively, and form the basis to understand their applications. The applications of chemicals will markedly progress due to emerging technologies, such as artificial intelligence for precision medicine, which is also true of the study of the estrogenome including estrogenic flavonoids.

Novel estrogen-responsive genes (ERGs) for the evaluation of estrogenic activity.

Estrogen action is mediated by various genes, including estrogen-responsive genes (ERGs). ERGs have been used as reporter-genes and markers for gene expression. Gene expression profiling using a set of ERGs has been used to examine statistically reliable transcriptomic assays such as DNA microarray assays and RNA sequencing (RNA-seq). However, the quality of ERGs has not been extensively examined. Here, we obtained a set of 300 ERGs that were newly identified by six sets of RNA-seq data from estrogen-treated and control human breast cancer MCF-7 cells. The ERGs exhibited statistical stability, which was based on the coefficient of variation (CV) analysis, correlation analysis, and examination of the functional association with estrogen action using database searches. A set of the top 30 genes based on CV ranking were further evaluated quantitatively by RT-PCR and qualitatively by a functional analysis using the GO and KEGG databases and by a mechanistic analysis to classify ERα/ß-dependent or ER-independent types of transcriptional regulation. The 30 ERGs were characterized according to (1) the enzymes, such as metabolic enzymes, proteases, and protein kinases, (2) the genes with specific cell functions, such as cell-signaling mediators, tumor-suppressors, and the roles in breast cancer, (3) the association with transcriptional regulation, and (4) estrogen-responsiveness. Therefore, the ERGs identified here represent various cell functions and cell signaling pathways, including estrogen signaling, and thus, may be useful to evaluate estrogenic activity.

A conserved regulatory element in the mammalian ß-globin promoters.

We provide here evidence for a conserved regulatory element for transcription of the ß-family globin genes based on a comparative study of 32 genes from 16 mammals. The element is characterized by the appearance of AA or TT dinucleotides in the A + T-rich region located 200-400 bp upstream of the cap sites. G-tracts 3-5 nucleotides long exist between the A + T-rich region and the conserved transcription factor binding sites (GATA-1 site and the CACCC, CCAAT, and ATA boxes) apparently dividing the regions. The average periodicity of AA or TT dinucleotides in the region from a total of 18 ß-family globin genes from four species was approximately 10 bp, suggesting that the DNA in these regions shows right-handed superhelicity. The proposed biological function of this element is to adjust the spatial positions for the first interaction of the transcription factor(s) which can recognize specific DNA sequences in the presence of packed chromatin.

Application of Fluolid-Orange-labeled probes for DNA microarray and immunological assays.

The usefulness of Fluolid-Orange, a novel fluorescent dye, for DNA microarray and immunological assays has been examined. Fluolid-Orange-labeled probes (DNA and IgG) were stable as examined by laser-photo-bleaching and under heat and dry conditions. Statistical analyses were performed to evaluate the reproducibility of the microarray assay, while stage-specific immunostaining of marker proteins, Kank1 and calretinin, was performed for renal cancers, both giving satisfactory results. The stability of the dye should provide advantages for storing fluorescently labeled probes and re-examining the specimens later in genetic and pathological diagnostics.

Nutritional implications of ginger: chemistry, biological activities and signaling pathways.

Ginger (Zingiber officinale Roscoe) has been used as a food, spice, supplement and flavoring agent and in traditional medicines due to its beneficial characteristics such as pungency, aroma, nutrients and pharmacological activity. Ginger and ginger extracts were reported to have numerous effects, such as those on diabetes and metabolic syndrome, cholesterol levels and lipid metabolism, and inflammation, revealed by epidemiological studies. To understand the beneficial characteristics of ginger, especially its physiological and pharmacological activities at the molecular level, the biological effects of ginger constituents, such as monoterpenes (cineole, citral, limonene and α/ß-pinenes), sesquiterpenes (ß-elemene, farnesene and zerumbone), phenolics (gingerols, [6]-shogaol, [6]-paradol and zingerone) and diarylheptanoids (curcumin), and the associated signaling pathways are summarized. Ginger constituents are involved in biological activities, such as apoptosis, cell cycle/DNA damage, chromatin/epigenetic regulation, cytoskeletal regulation and adhesion, immunology and inflammation, and neuroscience, and exert their effects through specific signaling pathways associated with cell functions/mechanisms such as autophagy, cellular metabolism, mitogen-activated protein kinase and other signaling, and development/differentiation. Estrogens, such as phytoestrogens, are one of the most important bioactive materials in nature, and the molecular mechanisms of estrogen actions and the assays to detect them have been discussed. The molecular mechanisms of estrogen actions induced by ginger constituents and related applications, such as the chemoprevention of cancers, and the improvement of menopausal syndromes, osteoporosis, endometriosis, prostatic hyperplasia, polycystic ovary syndrome and Alzheimer's disease, were summarized by a comprehensive search of references to understand more about their health benefits and associated health risks.

Kank proteins: a new family of ankyrin-repeat domain-containing proteins.

The human Kank gene was found as a candidate tumor suppressor for renal cell carcinoma, and encodes an ankyrin-repeat domain-containing protein, Kank. Here, we report a new family of proteins consisting of three Kank (Kank1)-associated members, Kank2, Kank3 and Kank4, which were found by domain and phylogenetic analyses. Besides the conserved ankyrin-repeat and coiled-coil domains, there was a conserved motif at the N-terminal (KN motif) containing potential motifs for nuclear localization and export signals. Gene expression of these genes was examined by RT-PCR at the mRNA level and by Western blotting and immunostaining at the protein level. Kank family genes showed variations in the expression level among tissues and kidney cell lines. Furthermore, the results of overexpression of these genes in NIH3T3 cells suggest that all of these family proteins have an identical role in the formation of actin stress fibers.

A major mutation of KIF21A associated with congenital fibrosis of the extraocular muscles type 1 (CFEOM1) enhances translocation of Kank1 to the membrane.

Congenital fibrosis of the extraocular muscles type 1 (CFEOM1) is associated with heterozygous mutations in the KIF21A gene, including a major (R954W) and a minor (M947T) mutation. Kank1, which regulates actin polymerization, cell migration and neurite outgrowth, interacted with the third and fourth coiled-coil domains of KIF21A protein at its ankyrin-repeat domain. While both KIF21A(R954W) and KIF21A(M947T) enhanced the formation of a heterodimer with the wild type, KIF21A(WT), these mutants also enhanced the interaction with Kank1. Knockdown of KIF21A resulted in Kank1 predominantly occurring in the cytosolic fraction, while KIF21A(WT) slightly enhanced the translocation of Kank1 to the membrane fraction. Moreover, KIF21A(R954W) significantly enhanced the translocation of Kank1 to the membrane fraction. These results suggest that KIF21A regulates the distribution of Kank1 and that KIF21A mutations associated with CFEOM1 enhanced the accumulation of Kank1 in the membrane fraction. This might cause an abrogation of neuronal development in cases of CFEOM1 through over-regulation of actin polymerization by Kank1.

Estrogenic Activity of Coffee Constituents.

Here, the constituents of coffee with estrogenic activity are summarized by a comprehensive literature search, and their mechanisms of action for their physiological effects are discussed at the molecular and cellular levels. The estrogenic activity of coffee constituents, such as acids, caramelized products, carbohydrates, lignin, minerals, nitrogenous compounds, oil (lipids), and others, such as volatile compounds, was first evaluated by activity assays, such as animal tests, cell assay, ligand-binding assay, protein assay, reporter-gene assay, transcription assay, and yeast two-hybrid assay. Second, the health benefits associated with the estrogenic coffee constituents, such as bone protection, cancer treatment/prevention, cardioprotection, neuroprotection, and the improvement of menopausal syndromes, were summarized, including their potential therapeutic/clinical applications. Inconsistent results regarding mixed estrogenic/anti-estrogenic/non-estrogenic or biphasic activity, and unbeneficial effects associated with the constituents, such as endocrine disruption, increase the complexity of the effects of estrogenic coffee constituents. However, as the increase of the knowledge about estrogenic cell signaling, such as the types of specific signaling pathways, selective modulations of cell signaling, signal crosstalk, and intercellular/intracellular networks, pathway-based assessment will become a more realistic means in the future to more reliably evaluate the beneficial applications of estrogenic coffee constituents.

Site-specific regulation of gene expression by estrogen in the hypothalamus of adult female rats.

Estrogen plays critical roles in the neuroendocrine system of adult female rats through separate actions, respectively, in the preoptic area (POA) and the ventromedial nucleus of the hypothalamus (VMH). Seven-week-old rats were treated with/without estrogen after they were ovariectomized, and four estrogen-responsive, neuronal system-related genes, encoding alpha4 neuronal nicotinic acetylcholine receptor (Chrna4), GABA(A) receptor delta (Gabrd), serotonin receptor 6 (Htr6), and GABA transporter 2 (Slc6a13), were investigated by real-time RT-PCR and Western blot analyses to examine their differential regulation by estrogen between the anterior part containing POA and the posterior part containing VMH. We further examined Bax, Bcl2, and Prkce, the former two genes to be involved in the gene expression network of Chrna4 and the latter gene, that of Gabrd. The regulation of Bax and Bcl2 by estrogen differed between the anterior and posterior parts. The results demonstrated differential regulation of these neuronal system-related genes by estrogen between the anterior and posterior parts of the hypothalamus and suggested the roles of gene expression networks for the respective genes in the neuroendocrine system of adult female rats.

Evaluation of estrogenic activity of phthalate esters by gene expression profiling using a focused microarray (EstrArray).

Phthalates are used industrially as plasticizers and are known to contaminate natural environments, mostly as di-ester or mono-ester complexes. Because they are structurally similar to natural estrogens, they could act as endocrine disruptors. Here, we used a DNA microarray containing estrogen responsive genes (EstrArray) to examine gene expression profiles in MCF-7 cells treated with 10 microM butylbenzyl phthalate (BBP), dibutyl phthalate (DBP), diethyl phthalate (DEP), and diisopropyl phthalate (DIP) along with the natural estrogen 17beta-estradiol ([E(2)], 10 nM). The profiles for phthalate esters and E(2) were examined by correlation analysis using correlation coefficients (r-values) and cluster analysis. We found that BBP showed the highest correlation with E(2) (r = 0.85), and DEP and DIP showed moderate r-values (r = 0.52 and r = 0.49, respectively). Dibutyl phthalate exhibited the lowest (but still significant) correlation with E(2) (r = 0.36). Furthermore, among the pairs of chemicals, DEP-DIP and DIP-DBP showed very high correlations (r = 0.90 and r = 0.80, respectively), and the other pairs showed moderate relationships, which reflected how structurally close they are to each other. The analysis of six functional groups of genes (enzymes, signaling, proliferation, transcription, transport, and others) indicated that the genes belonging to the enzyme, transcription, and other functional groups showed common responses to phthalate esters and E(2). Although the effect of BBP was similar to that of E(2), the other phthalate esters showed different types of effects. These results indicate that the structure of estrogenic chemicals is strongly related to their estrogenic activity and can be evaluated by appropriate grouping of the responsive genes by focused microarray analysis.

Molecular characterization of delta beta-thalassemia and hereditary persistence of fetal hemoglobin in the Indian population.

delta beta-Thalassemia (delta beta-thal) and hereditary persistence of fetal hemoglobin (HPFH) are heterogeneous disorders characterized by elevated levels of Hb F in adult life. The two disorders should not be considered as unambiguously separate entities but rather as a group of disorders with a variety of partially overlapping phenotypes. This study was undertaken to determine the hematological and molecular characteristics of high Hb F determinants among Indians. A gap-polymerase chain reaction (gap-PCR)-based approach was used for molecular characterization of high Hb F phenotypes. Fifty-five unrelated individuals were studied. The molecular findings were correlated with the hematological data. DNA analysis identified the deletion-inversion (G)gamma((A)gamma delta beta)(0)-thal in 15 cases (27%) and the HPFH-3 (Indian deletion) determinant in 26 cases (47.2%) and the Vietnamese/Chinese determinant (27 kb deletion) in five cases (9%), which is being reported for the first time from India; 16% (nine cases) of the samples remained uncharacterized. This study emphasizes that delta beta-thal and HPFH determinants are common in India. Molecular analysis will aid in understanding genotype-phenotype correlations and will facilitate prevention and control programs of thalassemia and hemoglobinopathies in this region.

Genome sequence of the cauliflower mushroom Sparassis crispa (Hanabiratake) and its association with beneficial usage.

Sparassis crispa (Hanabiratake) is a widely used medicinal mushroom in traditional Chinese medicine because it contains materials with pharmacological activity. Here, we report its 39.0-Mb genome, encoding 13,157 predicted genes, obtained using next-generation sequencing along with RNA-seq mapping data. A phylogenetic analysis by comparison with 25 other fungal genomes revealed that S. crispa diverged from Postia placenta, a brown-rot fungus, 94 million years ago. Several features specific to the genome were found, including the A-mating type locus with the predicted genes for HD1 and HD2 heterodomain transcription factors, the mitochondrial intermediate peptidase (MIP), and the B-mating type locus with seven potential pheromone receptor genes and three potential pheromone precursor genes. To evaluate the benefits of the extract and chemicals from S. crispa, we adopted two approaches: (1) characterization of carbohydrate-active enzyme (CAZyme) genes and ß-glucan synthase genes and the clusters of genes for the synthesis of second metabolites, such as terpenes, indoles and polyketides, and (2) identification of estrogenic activity in its mycelial extract. Two potential ß-glucan synthase genes, ScrFKS1 and ScrFKS2, corresponding to types I and II, respectively, characteristic of Agaricomycetes mushrooms, were newly identified by the search for regions homologous to the reported features of ß-glucan synthase genes; both contained the characteristic transmembrane regions and the regions homologous to the catalytic domain of the yeast ß-glucan synthase gene FKS1. Rapid estrogenic cell-signaling and DNA microarray-based transcriptome analyses revealed the presence of a new category of chemicals with estrogenic activity, silent estrogens, in the extract. The elucidation of the S. crispa genome and its genes will expand the potential of this organism for medicinal and pharmacological purposes.

Estrogen-responsive genes newly found to be modified by TCDD exposure in human cell lines and mouse systems.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) can induce estrogenic action or inhibit estrogen-induced effects in various tissues because of aryl hydrocarbon receptor (AhR)-estrogen receptor (ER) cross-talk. In order to identify the biomarkers of TCDD endocrine disruption, we screened estrogen-responsive genes modified by TCDD exposure using specific cDNA microarrays spotted with estrogen-responsive genes. MCF-7 human breast carcinoma cells and RL95-2 human endometrial carcinoma cells were exposed to TCDD, and an analysis of their gene expression revealed 32 genes exhibiting a significant change. The mRNA expression levels of 27 genes were subsequently verified using real-time RT-PCR. Among these genes, bioinformatic analyses indicated that insulin-like growth factor-binding protein 5 (IGFBP5) gene expression might be influenced by estrogen status. In our animal experiments, IGFBP5 was also shown to be responsive to TCDD exposure in mouse fetuses in utero. These results suggest that TCDD affects the expression levels of a series of estrogen-responsive genes, and follow-up fetal studies in mice indicated that IGFBP5 is useful as a biomarker of TCDD activity.